PROJECT SUMMERY/ABSTRACT: Neural crest cells, a multipotent stem cell-like population unique to vertebrate embryos, migrate great distances during development to form a wide variety of different cell types including craniofacial cartilage and bone, melanocytes, the adrenal medulla, and much of the peripheral nervous system. Defects in neural crest cell migration result in craniofacial maladies such as orofacial clefts, and dysregulation of neural crest derived cell types leads to a number of invasive cancers such as neuroblastoma. Recent work in the Gammill lab identified NSD3 as the first lysine methyltransferase that is essential for neural crest gene expression and migration. Although the known role of NSD3 is to dimethylate the 36th lysine residue of histone H3, depletion of NSD3 does not significantly alter H3K36me2 occupancy on neural crest genes even though these genes are no longer expressed. These results, as well as the cytoplasmic localization of NSD3 in migrating neural crest cells, the importance of cytoplasmic protein methylation during neural crest migration, and increasing evidence that ?histone? methyltransferases can methylate additional non-histone proteins, leads to the hypothesis that NSD3 methylates non-histone targets in order to regulate neural crest cell migration and gene expression. To explore this hypothesis, this proposal aims to define the essential role of NSD3 during cell migration in cell culture and in chick embryos by characterizing NSD3 non-histone targets and the migratory features regulated by their methylation. This will be accomplished by (1) defining the specific aspects of migration that are defective in NSD3 knockdown cells, (2) identifying non-histone proteins methylated by NSD3 through mass spectrometry-based quantitative proteomics, and (3) assaying the requirement for NSD3 non-histone targets during migration, and determining the importance of NSD3-dependent methylation for their function. Accomplishing these goals will define a novel mode of regulation governing neural crest cell migration and craniofacial development, and will reveal new functions and targets of NSD3. These results will reveal unappreciated post-translational control over cell migration, will uncover a novel regulatory mechanism in development, and will have important implications for efforts to prevent craniofacial birth defects and develop treatments for metastasis.